Pyridones and their use as modulators of serine hydrolase enzymes

ABSTRACT

This invention relates to a compound of formula I:                    
     or a pharmaceutically acceptable salt thereof; 
     in which preferably R 3 , R 4  and R 6  are each hydrogen; 
     X is C═O or CH 2 ; and 
     R 7  and R 8  are each independently selected from the group consisting of hydrogen, (C 1 -C 12 )alkyl, (C 3 -C 8 )cycloalkyl and (C 1 -C 12 )alkyl (C 6 -C 14 )aryl; or 
     R 7  and R 8  when taken together form a (C 2 -C 7 )alkylene group; or 
     —NR 7 R 8  together forms a (C 2 -C 14 )heterocyclic or substituted (C 2 -C 14 )heterocyclic. Such compounds modulate the activity of serine hydrolases and can be used in pharmaceutical compositions for the treatment of Alzheimer&#39;s disease.

RELATED APPLICATIONS

This Application is a division of U.S. Ser. No. 09/545,994, filed onApr. 10, 2000, the contents of which are hereby incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a common neurodegenerative disorder causingdementia. The incidence of AD increases with age (1). The prevalence ofdementia rises from 3% at age 65 years to 47% after age 85 years (1).The population of the elderly continues to rise and hence incidence ofAD is also expected to rise. The frequency of dementia doubles every 5years after the age of 60 years. In the United States, the annual costfor AD is estimated to be in excess of $60 billion annually (2, 3). Withthe rise in numbers of elderly individuals, the prevalence of AD is alsoexpected to rise with concomitant rise in the cost for AD. Developmentof drugs to delay the progression of AD as well as provide symptomatictreatment of this disorder is thus of paramount importance (1, 2, 3).

In AD there are three major microscopic features that are recognized asthe hallmarks of the disease, namely neuritic plaques (NP),neurofibrillary tangles. (NFT) and amyloid angiopathy (AA) (4). Inaddition, there is widespread cell loss, particularly of cholinergicneurons in the brain (5). Loss of cholinergic cells leads to reductionin the levels of the neurotransmitter acetylcholine, its synthesizingenzyme choline acetyltransferase, as well as its deactivating enzymeacetylcholinesterase (AChE) (5, 6). Reduction of cholinergicneurotransmission leads to some of the symptoms of AD (6).

Although the level of AChE is reduced in AD, the level of the closelyrelated enzyme butyrylcholinesterase (BuChE 3.1.1.8) is increased in ADbrains (7). BuChE is found in all the neuropathological lesionsassociated with AD, namely, NP, NFT and AA (7). Importantly, BuChE isfound in NP in brains of patients with AD. BuChE is found in a highernumber of plaques in brains of elderly individuals with AD relative tothose without AD (8). BuChE in Alzheimer brains requires 10-100 timesthe concentration of inhibitors to completely inhibit its esteraseactivity relative to BuChE in normal brains (9). It has been shown thatsome BuChE inhibitors not only improve cognition in an animal model butalso reduce the production of β-amyloid which is one of the principalconstituents of neuritic plaques (10).

From a neuropathology perspective, deposition of amyloid and formationof NP is one of the central mechanisms in the evolution of AD (11, 12).However, amyloid plaques are also found in brains of elderly individualswho do not have dementia (13). It has been suggested that the amyloidplaques in individuals without dementia are “benign” and they become“malignant”, causing dementia, when they are transformed intoplaques-containing degenerated neurites (13). These plaques are calledneuritic plaques (NP). The mechanism of transformation from “benign” to“malignant” plaques is as yet unknown. It has been suggested that BuChEmay play a major role in this-transformation based on the observationthat BuChE is found predominately in plaques that contain dystrophicneurites and not in plaques without dystrophic neurites (13).

Taken together these observations suggest that in brains of patientswith AD there is a significant alteration of the biochemical propertiesof BuChE that alters its normal regulatory role in the brain thuscontributing to the pathology of AD.

Recently, a brain specific serine protease called trypsin IV has beenisolated and it is presumed to be involved in APP processing (24).Amyloid precursor protein (APP) is a transmembrane glycoprotein, whichpossesses a Kunitz-type serine protease inhibitor domain. The APP may beinvolved in protease regulation in the brain (14, 15). Of particularimportance is the fact that abnormally cleaved APP results in theformation of a 40-42 amino acid residue β-amyloid protein fragment. Thisfragment is the main constituent of NP (16).

The proteolytic sites in APP have been studied extensively (18). Thereare three known proteolytic sites. The first is the α-secretase sitewhich when cleaved yields a 120 KDa fragment that does not accumulate inamyloid plaques (18). A basic amino acid residue such as arginine atthis site is required for cleavage (19). Enzymes that require a basicamino acid residue at the cleavage site of their substrates are serinepeptidases, such as trypsin. The second cleavage site, the γ-secretasesite, cleaves at lys-28 (also a tryptic-site), which is the last aminoacid of the extracellular APP domain (20). The third cleavage site, theβ-secretase site, occurs at the N-terminus (21). The latter two siteslead to fragments that accumulate in amyloid plaques.

The enzymes that cleave amyloid precursor protein are called“secretases” but they have not been fully identified. (22). It has beenobserved that a basic amino acid residue is required at some of thesites where APP undergoes proteolytic cleavage (19). Two well-knownenzymes that cleave peptides at basic amino acid residue sites aretrypsin and carboxypeptidase B (23). Both of these enzymes are mainlyrecognized as pancreatic enzymes involved in digestion, but trypsin-likeserine proteases have been found in the brain and are thought to beinvolved in APP processing (24, 25, 26, 27). Interestingly, an enzymewith tryptic-like activity is closely associated with BuChE (28, 29).Recent observations that BuChE considerably enhances tryptic activityunder normal circumstances (30, 31) and the observations that BuChE,which is found in high levels in NP, has altered biochemical properties,suggests that there may be a loss of regulation of tryptic activity, andother serine peptidase activity, associated with BuChE. This loss ofregulation may play a role in abnormal proteolytic processing of APP.Recent evidence suggests that inhibition of BuChE enhances cognitiveperformance in rats, and that it promotes non-amyloidogenic processingof amyloid precursor protein (10).

Development of molecules that inhibit the activity of BuChE and/or AChEand simultaneously enhance the activity of serine proteases would notonly provide symptomatic treatment of AD but would also lead todiscovery of drugs that stop the progression of AD.

SUMMARY OF THE INVENTION

The present invention provides 2-pyridones that modulate serinehydrolase activity. They inhibit activity of BuChE and or AChE andstimulate activity of trypsin.

More specifically, the present invention provides a compound of formulaI:

or a pharmaceutically acceptable salt thereof;

wherein X is C═O, C═S or CH₂;

R³, R⁴ and R⁶ are each independently selected from the group consistingof hydrogen, (C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl,(C₃-C₈)cycloalkyl, substituted (C₃-C₈)cycloalkyl, (C₂-C₁₂)alkenyl,substituted (C₂-C₁₂)alkenyl, (C₂-C₁₂)alkynyl, substituted(C₂-C₁₂)alkynyl, (C₆-C₁₄)aryl, substituted (C₆-C₁₄)aryl, (C₁-C₁₂)alkyl(C₆-C₁₄)aryl, substituted (C₁-C₁₂)alkyl (C₆-C₁₄)aryl, (C₆-C₁₄)aryl(C₁-C₁₂)alkyl, substituted (C₆-C₁₄)aryl (C₁-C₁₂)alkyl, (C₆-C₁₄)aryl(C₂-C₁₂)alkenyl, substituted (C₆-C₁₄)aryl (C₂-C₁₂)alkenyl, (C₆-C₁₄)aryl(C₂-C₁₂)alkynyl, substituted (C₆-C₁₄)aryl (C₂-C₁₂)alkynyl,(C₂-C₁₄)heterocyclic, substituted (C₂-C₁₄)heterocyclic, trifluoromethyl,halogen, cyano and nitro;

—S(O)R′, —S(O)₂R′, —S(O)₂OR′ and —S(O)₂NHR′, wherein each R′ isindependently (C₁-C₁₂)alkyl, (C₂-C₁₂)alkenyl, (C₂-C₁₂)alkynyl or(C₆-C₁₄)aryl;

—C(O)R″, wherein R″ is selected from the group consisting of hydrogen,(C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl, (C₃-C₈)cycloalkyl, substituted(C₃-C₈)cycloalkyl, (C₁-C₁₂)alkoxy, (C₁-C₁₂)alkylamino, (C₂-C₁₂)alkenyl,substituted (C₂-C₁₂)alkenyl, (C₂-C₁₂)alkynyl, substituted(C₂-C₁₂)alkynyl, (C₆-C₁₄)aryl, substituted (C₆-C₁₄)aryl,(C₆-C₁₄)aryloxy, (C₆-C₁₄)arylamino, (C₁-C₁₂)alkyl (C₆-C₁₄)aryl,substituted (C₁-C₁₂)alkyl (C₆-C₁₄)aryl, (C₆-C₁₄)aryl (C₁-C₁₂)alkyl,substituted (C₆-C₁₄)aryl (C₁-C₁₂)alkyl, (C₆-C₁₄)aryl (C₂-C₁₂)alkenyl,substituted (C₆-C₁₄)aryl (C₂-C₁₂)alkenyl, (C₆-C₁₄)aryl (C₂-C₁₂)alkynyl,substituted (C₆-C₁₄)aryl (C₂-C₁₂)alkynyl, (C₂-C₁₄)heterocyclic,substituted (C₂-C₁₄)heterocyclic and trifluoromethyl;

—OR′″ and —NR′″₂, wherein each R′″ is independently selected fromhydrogen, (C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl, (C₃-C₈)cycloalkyl,substituted (C₃-C₈)cycloalkyl, (C₂-C₁₂)alkenyl, substituted(C₂-C₁₂)alkenyl, (C₂-C₁₂)alkynyl, substituted (C₂-C₁₂)alkynyl,(C₆-C₁₄)aryl, substituted (C₆-C₁₄)aryl, (C₁-C₁₂)alkyl (C₆-C₁₄)aryl,substituted (C₁-C₁₂)alkyl (C₆-C₁₄)aryl, (C₆-C₁₄)aryl (C₁-C₁₂)alkyl,substituted (C₆-C₁₄)aryl (C₁-C₁₂)alkyl, (C₆-C₁₄)aryl (C₂-C₁₂)alkenyl,substituted (C₆-C₁₄)aryl (C₂-C₁₂)alkenyl, (C₆-C₁₄)aryl (C₂-C₁₂)alkynyl,substituted (C₆-C₁₄)aryl (C₂-C₁₂)alkynyl, (C₆-C₁₄)aroyl, substituted(C₆-C₁₄)aroyl, (C₂-C₁₄)heterocyclic, substituted (C₂-C₁₄)heterocyclic,(C₁-C₁₂)acyl and trifluoromethyl;

—SR″″, wherein R″″ is selected from the group consisting of hydrogen,(C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl, (C₂-C₁₂)alkenyl, substituted(C₂-C₁₂)alkenyl, (C₂-C₁₂)alkynyl, substituted (C₂-C₁₂)alkynyl,(C₆-C₁₄)aryl, substituted (C₆-C₁₄)aryl, (C₁-C₁₂)alkyl (C₆-C₁₄)aryl,substituted (C₁-C₁₂)alkyl (C₆-C₁₄)aryl, (C₆-C₁₄)aryl (C₁-C₁₂)alkyl,substituted (C₆-C₁₄)aryl (C₁-C₁₂)alkyl, (C₆-C₁₄)aryl (C₂-C₁₂)alkenyl,substituted (C₆-C₁₄)aryl (C₂-C₁₂)alkenyl, (C₆-C₁₄)aryl (C₂-C₁₂)alkynyl,substituted (C₆-C₁₄)aryl (C₂-C₁₂)alkynyl, (C₂-C₁₄)heterocyclic,substituted (C₂-C₁₄)heterocyclic and trifluoromethyl; and

—SiR′″″₃, wherein R′″″ is selected from (C₁-C₁₂)alkyl or (C₆-C₁₄)aryl;and

R⁷ and R⁸ are each independently selected from the group consisting ofhydrogen, (C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl, (C₃-C₈)cycloalkyl,substituted (C₃-C₈)cycloalkyl, (C₂-C₁₂)alkenyl, substituted(C₂-C₁₂)alkenyl, (C₂-C₁₂)alkynyl, substituted (C₂-C₁₂)alkynyl,(C₆-C₁₄)aryl, substituted (C₆-C₁₄)aryl, (C₁-C₁₂)alkyl (C₆-C₁₄)aryl,substituted (C₁-C₁₂)alkyl (C₆-C₁₄)aryl, (C₆-C₁₄)aryl (C₁-C₁₂)alkyl,substituted (C₆-C₁₄)aryl (C₁-C₁₂)alkyl, (C₆-C₁₄)aryl (C₂-C₁₂)alkenyl,substituted (C₆-C₁₄)aryl (C₂-C₁₂)alkenyl, (C₆-C₁₄)aryl (C₂-C₁₂)alkynyl,substituted (C₆-C₁₄)aryl (C₂-C₁₂)alkynyl, (C₂-C₁₄)heterocyclic,substituted (C₂-C₁₄)heterocyclic and trifluoromethyl; or

—NR⁷R⁸ forms a (C₂-C₁₄)heterocyclic or substituted (C₂-C₁₄)heterocyclicgroup;

wherein the substituted groups listed above are substituted with one ormore substituents selected from the group consisting of hydroxy,(C₁-C₄)alkoxy, (C₆-C₁₄)aryl, (C₂-C₁₄)heterocyclic, halogen,trifluoromethyl, cyano, nitro, amino, carboxyl, carbamate, sulfonyl andsulfonamide,; and

the heterocyclic group contains at least one atom, preferably two,selected from oxygen, nitrogen and sulfur.

The present invention also provides a pharmaceutical compositioncomprising a compound of formula I disclosed herein, or apharmaceutically acceptable salt thereof, together with apharmaceutically acceptable diluent or carrier. Preferably thepharmaceutical composition of the invention is for the modulation of anactivity of a serine hydrolase.

Compounds of the formula I, while depicted herein in their “keto”tautomeric form, can also exist in their corresponding “enol” tautomericform.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a plot of absorbance per minute against the log ofconcentration of 5-(N,N-dibenzyl)aminocarbonyl-2-pyridone. The graphreflects the rate of hydrolysis of the substrate (acetylthiocholine(AcSCh) for AChE and butyrylthiocholine (BuSCh) for BuChE).

FIG. 2 is a plot of absorbance per minute against the log ofconcentration of 5-(N,N-diisopropyl)aminocarbonyl-2-pyridone. The graphreflects the rate of hydrolysis of the substrate (acetylthiocholine forAChE and butyrylthiocholine for BuChE).

FIG. 3 is a plot of absorbance per minute against the log ofconcentration of 5-(N,N-diethyl)aminocarbonyl-2-pyridone. The graphreflects the rate of hydrolysis of the substrate (acetylthiocholine forAChE and butyrylthiocholine for BuChE).

FIG. 4 is a plot of absorbance per minute against the log ofconcentration of 5-(N,N-diethyl)aminomethyl-2-pyridone. The graphreflects the rate of hydrolysis of the substrate (acetylthiocholine forAChE and butyrylthiocholine for BuChE).

FIG. 5 is a plot of absorbance per minute against the log ofconcentration of 5-(1-pyrrolidinyl)carbonyl-2-pyridone. The graphreflects the rate of hydrolysis of the substrate (acetylthiocholine forAChE and butyrylthiocholine for BuChE).

FIG. 6 is a plot of absorbance per minute against the log ofconcentration of 5-(1-piperidinyl)carbonyl-2-pyridone. The graphreflects the rate of hydrolysis of the substrate (acetylthiocholine forAChE and butyrylthiochdline for BuChE).

FIG. 7 is a plot of absorbance per minute against the log ofconcentration of 5-(N-cyclohexyl)aminocarbonyl-2-pyridone. The graphreflects the rate of hydrolysis of the substrate butyrylthiocholine byBuChE. There was no inhibition of the activity of AChE on its substrateacetylthiocholine (data not shown).

FIG. 8 is a plot of absorbance per minute against the log ofconcentration of 5-(N-phenothiazinyl)carbonyl-2-pyridone. The graphreflects the rate of hydrolysis of the substrate (acetylthiocholine forAChE and butyrylthiocholine for BuChE).

FIG. 9 is a plot of absorbance per minute against the log ofconcentration of 5-(N-phenoxazinyl)carbonyl-2-pyridone. The graphreflects the rate of hydrolysis of the substrate (acetylthiocholine forAChE and butyrylthiocholine for BuChE).

FIG. 10 is a plot of absorbance per minute against the log ofconcentration of 5-(N-(N-methyl)piperazinyl)aminocarbonyl-2-pyridone.The graph reflects the rate of hydrolysis of the substrate(acetylthiocholine for AChE and butyrylthiocholine for BuChE).

FIG. 11 is a bar diagram that shows the effect of the compounds5-(N,N-dibenzyl)aminocarbonyl-2-pyridone (Example 1) and5-(N-phenothiazinyl)carbonyl-2-pyridone (Example 8) on the trypsin-likeactivity associated with BuChE. The first bar in this figure, labeled“No”, is the activity of the enzyme with trypsin-like activityassociated with BuChE in the absence of any added compound.

FIG. 12 is a bar diagram showing the effect of5-(N-phenothiazinyl)carbonyl-2-pyridone (Example 8) on trypsin activity.The first bar in this figure, labeled “No”, is the activity of trypsinin the absence of any added compound.

DETAILED DESCRIPTION OF THE INVENTION

As employed herein, “lower alkyl” refers to straight or branched chainalkyl groups having 1 to 4 carbon atoms;

“alkyl” refers to straight or branched chain alkyl groups having 1 to 12carbon atoms;

“substituted alkyl” refers to alkyl groups bearing one or moresubstituents such as hydroxy, (C₁-C₄)alkoxy, (C₆-C₁₄)aryl,(C₂-C₁₄)heterocyclic, halogen, trifluoromethyl, cyano, nitro, amino,carboxyl, carbamate, sulfonyl, sulfonamide, and the like;

“cycloalkyl” refers to cyclic ring-containing groups containing 3 to 8carbon atoms, and “substituted cycloalkyl” refers to cycloalkyl groupsbearing one or more substituents as set forth above;

“alkenyl” refers to straight or branched chain hydrocarbyl groups havingat least one carbon-carbon double bond, and having 2 to 12 carbon atoms(with groups having 2 to 6 carbon atoms presently being preferred), and“substituted alkenyl” refers to alkenyl groups bearing one or moresubstituents as set forth above;

“alkynyl” refers to straight or branched chain hydrocarbyl groups havingat least one carbon-carbon triple bond, and having 2 to 12 carbon atoms(with groups having 2 to 6 carbon atoms presently being preferred), and

“substituted alkynyl” refers to alkynyl groups bearing one or moresubstituents as set forth above;

“aryl” refers to aromatic groups having 6 to 14 carbon atoms and“substituted aryl” refers to aryl groups bearing one or moresubstituents as set forth above;

“alkylaryl” refers to alkyl-substituted aryl groups and “substitutedalkylaryl” refers to alkylaryl groups bearing one or more substituentsas set forth above;

“arylalkyl” refers to aryl-substituted alkyl groups and “substitutedarylalkyl” refers to arylalkyl groups bearing one or more substituentsas set forth above;

“arylalkenyl” refers to aryl-substituted alkenyl groups and “substitutedarylalkenyl” refers to arylalkenyl groups bearing one or moresubstituents as set forth above;

“arylalkynyl” refers to aryl-substituted alkynyl groups and “substitutedarylalkynyl” refers to arylalkynyl groups bearing one or moresubstituents as set forth above;

“aroyl” refers to aryl-carbonyl species such as benzoyl and “substitutedaroyl” refers to aroyl groups bearing one or more substituents as setforth above;

“heterocyclic” refers to cyclic (i.e., ring containing) groupscontaining one or more heteroatoms (e.g., N, O, S, or the like) as partof the ring structure, and having 2 to 14 carbon atoms and “substitutedheterocyclic” refers to heterocyclic groups bearing one or moresubstituents as set forth above;

“acyl” refers to alkyl-carbonyl species; and

“halogen” refers to fluoride, chloride, bromide or iodide groups.

In preferred embodiments of the invention, R³, R⁴ and R⁶ are eachhydrogen.

In further preferred embodiments of the invention, X is C═O or CH₂.

In further preferred embodiments of the invention R⁷ and R⁸ are eachindependently selected from the group consisting of hydrogen,(C₁-C₁₂)alkyl, (C₃-C₈)cycloalkyl and (C₁-C₁₂)alkyl (C₆-C₁₄)aryl; or

—NR⁷R⁸ together forms a (C₂-C₁₄)heterocyclic or substituted(C₂-C₁₄)heterocyclic group. Preferably the heterocyclic or substitutedheterocyclic group includes a further heteroatom selected from nitrogen,sulfur and oxygen, and more preferably includes one or more fused benzogroups.

Also preferred are compounds in which, in —NR⁷R⁸, R⁷ and R⁸ togetherform a (C₂-C₇)alkylene group.

More preferred is a compound selected from the group consisting of:

5-(N,N-dibenzyl)aminocarbonyl-2-pyridone;

5-(N,N-diisopropyl)aminocarbonyl-2-pyridone;

5-(N,N-diethyl)aminocarbonyl-2-pyridone;

5-(N,N-diethyl)aminomethyl-2-pyridone;

5-(1-pyrrolidinyl)aminocarbonyl-2-pyridone;

5-(1-piperidinyl)aminocarbonyl-2-pyridone;

5-(N-cyclohexyl)aminocarbonyl-2-pyridone;

5-(N-phenothiazinyl)aminocarbonyl-2-pyridone;

5-(N-phenoxazinyl)aminocarbonyl-2-pyridone; and

5-(N-(N-methyl)piperazinyl)aminocarbonyl-2-pyridone.

The compounds of the invention modulate serine hydrolase activity.

Certain compounds of the invention are effective as inhibitors ofcholinesterases, for example butyrylcholinesterase (BuChE) andacetylcholinesterase (AChE).

Certain compounds of the invention are effective in enhancing theactivity of serine proteases, for example trypsin and a trypsin-likeprotein associated with BuChE in a brain of a mammal, such as a human.

The compounds of the invention can be used to treat, inhibit or preventa pathological condition that is manifested in an abnormal concentrationof, and/or activity of, a serine hydrolase enzyme. Among thosepathological conditions are Alzheimer's disease, tumours such as braintumours, for example gliomas, and glaucoma.

MATERIALS AND METHODS Synthesis of 2-Pyridone Compounds

The synthesis of exemplified 2-pyridone compounds was achieved insignificant yield in a two-step, one pot procedure.

Amides

Scheme I shows a method of preparing amide compounds of the invention inwhich R³, R⁴ and R⁶ are hydrogen. 6-hydroxy nicotinic acid is a readilyavailable starting material. It can be converted to the correspondingacid chloride with thionyl chloride which in turn can be used tosynthesize a variety of substituted amides of the general formula I insignificant yield.

Amines

Scheme II shows a method of preparing preferred amine compounds of theinvention in which R³, R⁴ and R⁶ are hydrogen. The acid chloride of6-hydroxy-nicotinic acid can be treated with methanol to give thecorresponding methyl ester, which can be reduced with lithium aluminumhydride to the corresponding 5-hydroxy methyl 2-pyridone. This can beconverted to the corresponding bromide with hydrobromic acid. The5-bromo methyl-2-pyridone can then be used to synthesize a variety ofsubstituted amines of the general formula I in significant yield.

EXAMPLE 1 5-(N,N-Dibenzyl)aminocarbonyl-2-pyridone (Tautomer FormN,N-Dibenzyl 6-Hydroxynicotinamide)

N,N-Dibenzyl 6-hydroxynicotinamide was made according to the aboveprocedure using 1.58 g (10.0 mmol) of 6-hydroxynicotinyl chloride and2.3 ml (12.0 mmol) of dibenzylamine to furnish 1.84 g (58%) of product.¹H NMR (CDCl₃, 200 MHz) δ: 7.73 (d, J=3.0 Hz, 1H), 7.65 (dd, J=9.0, 4.5Hz, 1H), 7.45-7.13 (m, 11H), 6.51 (d, J=9.0 Hz, 1H), 4.57 (bs, 4H). IR(CHCl₃) cm⁻¹: 3387, 3011, 1681, 1660, 1633, 1223. HREIMS m+/z (%):C₂₀H₁₈N₂O₂ (calc)=318.1369; C₂₀H₁₈N₂O₂ (obs)=318.1359 (90).

EXAMPLE 2 5-(N,N-Diisopropyl)aminocarbonyl-2-pyridone

A 200 ml round bottomed flask (RBF) was charged with 1.13 g (7.18 mmol)of 6-hydroxynicotinyl chloride in 100 ml of methylene chloride, cooledand stirred at 0° C. N,N-diisopropylamine (8.6 mM=0.85 ml) in 10 mlmethylene chloride was added drop wise and the resulting mixture wasstirred at room temperature for 15 hours. The mixture was thenconcentrated under vacuum. To the residue was added 25 ml of methylenechloride and stirred at 30° C. for 5 minutes. Solid was filtered andresidue was chromatographed using CH₂Cl₂:MeOH:NH₃=200:10:1. The productwas recrystallized from CH₂Cl₂ and petroleum ether (yield 29%). ¹H NMR(CD₃OD, 400 MHz, ppm): 7.58 (s, 1H), 7.56 (dd, J=2.6, 9 Hz, 1H), 6.57(dd, J=1.0, 9.06 Hz, 1H), 5.21 (bs, 1H), 3.80 (bs, 2H), 1.34 (m, 12H).¹³C NMR (CD₃OD, 400 MHz, ppm): 167.8, 163.6, 140.0, 134.2, 119.4, 117.8,49.0, 19.7. IR (CHCl₃) cm⁻¹: 3371, 3118, 2918, 2852, 1600, 1433, 1366,1335, 1128, 1090, 882, 597. HREIMS m+/z (%): C₁₂H₁₈N₂O₂ (calc)=222.1368,C₁₂H₁₈N₂O₂ (obs)=222.1362 (100).

EXAMPLE 3 5-(N,N-Diethyl)aminocarbonyl-2-pyridone

5-(N,N-diethyl)aminocarbonyl-2-pyridone was synthesized according to thegeneral procedure outlined above. Briefly, 1.58 g (10.0 mmol) of6-hydroxynicotinyl chloride was reacted with 2.07 ml (20.0 mmol) ofdiethylamine to furnish 0.87 g of the product. ¹H NMR (DMSO-d₆, 400 MHz)δ: 11.60 (bs, 1H), 7.47 (s, 1H), 7.43 (d, J=9.4 Hz, 1H), 6.32 (d, J=9.4Hz, 1H), 3.40-3.31 (bq, 6.7 Hz, 4H), 1.09 (t, J=6.7 Hz, 6H). ¹³C NMR(DMSO-d₆, 400 MHz) δ: 166.7, 161.6, 139.4, 135.3, 119.1, 114.3, 40.9,40.1, 13.2. IR (CHCl₃) cm⁻¹: 3370, 3120, 2915, 2860, 1605, 1430, 1366,1335, 1135, 1080, 882, 605. HREIMS m+/z (%): C₁₀H₁₄N₂O₂ (calc)=194.1056;C₁₀H₁₄N₂O₂ (obs)=194.1055 (100).

EXAMPLE 4 5-(N,N-Diethyl)aminomethyl-2-pyridone

6-hydroxy nicotinic acid (2 gm, 14.4 mmol) was mixed with 4.6 ml ofthionyl chloride and refluxed until clear and the mixture was maintainedat 80° C. for 20 min. The excess thionyl chloride was evaporated invacuo. The cooled 6-hydroxynicotinyl chloride was treated with 10 ml ofmethanol and the solution was refluxed for 1 hour. The excess methanolwas evaporated and the methyl 6-hydroxy nicotinate was crystallized fromacetone. The yield of the product was 1.88 gm (85%). ¹H NMR (d₆-DMSO,ppm); 11.8 (broad, H, NH), 8.03 (d, J=2.7 Hz, 1H, C(6)H), 7.78 (dd,J₃₋₄=9.6, J₄₋₆=2.7 Hz, 1H, C(4)H), 6.35 (d, J=9.6 Hz, 1H, C(3)H), 3.75(s, 3H, CH₃O).

To a suspension of LiAlH₄ (0.32 gm, 8.4 mmol) in 80 ml THF was added,slowly and dropwise, a solution of methyl 6-hydroxynicotinate (1.15 gm,7.5 mmol) in 400 ml THF. The mixture was stirred at room temperature for1.5 hours and then refluxed for 10 minutes. The mixture was then cooledand the reaction quenched with 3.0 ml of ethyl acetate and 1.5 ml ofwater. The solvents were removed and the residue was taken up in 40 mlof refluxing ethanol. The solution was filtered through celite andethanol was evaporated in vacuo. The product was purified by silica gelchromatography using ethyl acetate/methanol (2:1) as the eluent. Theproduct 5-hydroxymethyl-2-pyridone was crystallized from ethanol/ethylacetate and the yield of the reaction was 0.65 gm (80%). HRMS: m/e, M⁺found 125.0468, 100%. Calc. for C₆H₇NO₂: 125.0477, −6 ppm. ¹H NMR(d₆-DMSO): 11.47 (broad, 1H, NH), 7.39 (dd, J₃₋₄=9.5 Hz, J₄₋₆=2.5 Hz,1H, C(4)H), 7.23 (d, J₄₋₆=2.5 Hz, 1H, C(6)H), 6.27 (d, J₃₋₄=9.5 Hz, 1H,C(3)H), 5.10 (t, J=5.5 Hz, 1H, CH₂OH), 4.17 (d, J=5.5 Hz, 2H, CH₂OH). IR(cm⁻¹): 3271 (m, broad), υ (O—H); 3124 (m, broad), υ (N—H); 3011 (m), υ(C—H); 1661 (vs), υ (C═O).

To 5-hydroxymethyl-2-pyridone-(114.9 mg, 0.9 mmol) was added 3.0 ml of48% hydrobromic acid. The mixture was heated at 100° C. for 20 minutes.The excess hydrobromic acid was then evaporated in vacuo to give thecorresponding 5-bromomethyl-2-pyridone. This compound was used withoutpurification. HRMS: m/e, M+ found; 186.9626, 7.4% Calc. for C₆H₆BrNO:186.9633, −3.9 ppm.

The 5-bromomethyl-2-pyridone was taken up in diethyl amine and thesolution refluxed for 1 hour. The solution was then treated with 10%sodium hydroxide at 0° C. and washed with chloroform. The aqueous phasewas treated with hydrochloric acid to a pH of 6.0 and extracted withchloroform/methanol (5:1). The solution was dried over Na₂SO₄ and thesolvent was then evaporated. The product,(N,N-diethylaminomethyl)-2-pyridone, was obtained in 34% yield. HRMS:m/e, M+ found; 180.1251, 20.8%. Calc. for C₁₀H₁₆N₂O; 180.1263, −6.3 ppm.¹H NMR (ppm): 13.1 (broad, 1H, NH), 7.49 (dd, J₃₋₄=9.2 Hz, J₄₋₆=2.4 Hz,1H, C(4)H), 7.25 (d, J₄₋₆=2.4 Hz, 1H, C(6)H), 6.55 (d, J₃₋₄=9.2 Hz, 1H,C(3)H), 3.30 (s, 2H, CH₂O), 2.45 (q, J=7.1 Hz, 4H, N (CH₂CH₃)₂), 1.00(t, J=7.1 Hz, 6H, N (CH₂CH₃)₂). IR (cm⁻¹): 1660 (vs), υ (C═O).

EXAMPLE 5 5-(1-Pyrrolidinyl)aminocarbonyl-2-pyridone

In a 250 ml RBF was put 2.6 g of 6-hydroxynicotinic acid and to this wasadded 25 ml of SOCl₂. The flask was then equipped with a refluxcondenser and a CaCl₂ drying tube. The slurry was brought to reflux andafter 30-45 minutes the solution became homogenous. The solution wasthen refluxed for an additional 15 minutes and then the SOCl₂ wasimmediately evaporated in vacuo and then put on a vacuum pump for halfan hour. The solid was then taken up in CH₂Cl₂ (225 mls) and 1.9 mls offreshly distilled pyrrolidine was added dropwise over 2 minutes. Thesolution was then stirred at room temperature for 16 hours under aninert atmosphere. The reaction mixture was concentrated to approximately75 mls and then was filtered through a pad of celite. The filtrate wasevaporated to dryness to give a tan colored glass. The solid was takenup in CH₂Cl₂ ((50 mls) and washed with 1 M NaOH (4×25 mls). Combinedaqueous phase was acidified by addition of 10 mls of conc. HCl and thenextracted with n-BuOH (4×25 mls). The combined organic phase was washedwith saline (1×) and then dried (MgSO4), filtered and solvent evaporatedto give a tan colored solid. Purified on SiO2 (10:1 CH₂Cl₂/MeOH aseluent) and crystallized from CH₂Cl₂/petroleum ether to furnish 1.08 g(56%) of product. ¹H NMR (CDCl₃, 400 MHz) δ: 7.71 (s, 1H), 7.63 (d,J=8.5 Hz, 1H), 6.44 (d, J=8.6 Hz, 1H), 3.45 (m, 4H), 1.82 (m, 4H). ¹³CNMR (CDCl₃, 400 MHz) d: 165.4, 164.5, 140.8, 136.4, 119.4, 116.3, 49.5,46.7, 26.5, 24.1. IR (CHCl₃) cm⁻¹: 3365, 3113, 2905, 2823, 1650, 1570,1483, 1335, 1120, 827, 608. HREIMS: m+/z (%): C₁₀H₁₂N₂O₂(calc)=192.0900; C₁₀H₁₂N₂O₂ (obs)=192.0892 (85).

EXAMPLE 6 5-(1-Piperidinyl)aminocarbonyl-2-pyridone (Tautomer FormPiperidinyl-6-hydroxynicotinamide)

A 100 ml RBF was charged with 6-hydroxynicotinic acid (4 g) and 20 ml ofthionyl chloride. The mixture was refluxed for 1 hr. Excess thionylchloride was then evaporated in vacuo to obtain the 6-hydroxynicotinylchloride. A 200 ml RBF was charged with 1.13 g of 6-hydroxynicotinylchloride in 100 ml of methylene chloride, cooled and stirred at 0° C.Piperidine (8.6 mM=0.85 ml) in 10 ml methylene chloride was addeddropwise and the resulting mixture was stirred at room temperature for15 hours. The mixture was then concentrated under vacuum. To the residuewas added 25 ml of methylene chloride and stirred at 30° C. for 5minutes. Solid was filtered and residue was chromatographed usingCH₂Cl₂:MeOH:NH₃=200:10:1. The product was recrystallized from CH₂Cl₂ andpetroleum ether to furnish 0.92 g (62%) of product. ¹H NMR (CDCl₃, 400MHz) δ: 7.61 (s, 1H), 7.55 (d, J=8.4 Hz, 1H), 6.55 (d, J=8.6 Hz, 1H),3.50 (bs, 4H), 1.65 (m, 2H), 1.57 (m, 4H). ¹³C NMR (CDCl₃, 400 MHz) δ:166.5, 164.8, 141.3, 135.7, 119.7, 116.1, 26.0, 24.4. IR (CHCl₃) cm⁻¹:3346, 3050, 3004, 1655, 1615, 1473, 1115, 841, 780, 627. HREIMS m+/z(%): C₁₁H₁₄N₂O₂ (calc)=206.1055; C₁₁H₁₄N₂O₂ (obs)=206.1061 (70).

EXAMPLE 7 5-(N-Cyclohexyl)aminocarbonyl-2-pyridone (Tautomer FormN-Cyclohexyl 6-Hydroxynicotinamide)

N-Cyclohexyl 6-hydroxynicotinamide was synthesized according to thegeneral procedure using 1.58 g (10.0 mmol) of 6-hydroxynicotinylchloride and 1.37 ml (12.0 mmol) of cyclohexylamine to furnish 1.54 g(70%) of the product. ¹H NMR (CD₃OD, 400 MHz) δ: 8.03 (s, 1H), 7.97 (d,J=8.4 Hz, 1H), 6.52 (d, J=8.6 Hz, 1H), 3.98 (m, 4H), 1.92 (m, 2H), 1.78(m, 2H), 1.68 (m, 1H), 1.32 (m, 5 H). ¹³C NMR (CD₃OD, 400 MHz) δ: 164.2,164.1, 139.7, 136.8, 118.6, 114.6, 49.1, 48.2, 46.9, 32.3, 25.2, 25.0.IR (Nujol) cm⁻¹: 3294, 3062, 1637, 1545. HREIMS m+/z (%): C₁₂H₁₆N₂O₂(calc)=220.1212; C₁₂H₁₆N₂O₂ (obs)=220.1205 (100).

EXAMPLE 8 5(N-Phenothiazinyl)aminocarbonyl-2-pyridone (Tautomer FormPhenothiazinyl 6-Hydroxynicotinamide)

Phenothiazinyl 6-hydroxynicotinamide was synthesized according to thegeneral procedure using 1.2 g (7.6 mmol) of 6-hydroxynicotinyl chlorideand 2.93 g (14.7 mmol) of phenothiazine to furnish 0.71 g (31%) ofproduct. ¹H NMR (DMSO-d₆, 400 MHz) δ: 11.85 (bs, 1H), 7.62-7.57 (m, 4H),7.44 (d, J=2.6 Hz, 1H), 7.35-7.25 (m, 4H), 7.05 (dd, J=9.8, 2.7 Hz, 1H),6.12 (d, J=9.6 Hz, 1H). ¹³C NMR (DMSO-d₆, 400 MHz) δ: 164.9, 161.9,140.2, 139.4, 131.7, 128.3, 127.8, 127.3, 127.3, 119.0, 112.9. IR (KBr)cm⁻¹: 3446, 3070, 3054, 1645, 1615, 1460, 1351, 1266, 1114, 841, 780,627. HREIMS m+/z (%): C₁₈H₁₂N₂O₂S (calc)=320.0621; C₁₈H₁₂N₂O₂S(obs)=320.0622 (100).

EXAMPLE 9 5-(N-Phenoxazinyl)aminocarbonyl-2-pyridone (Tautomer FormPhenoxazinyl 6-Hydroxynicotinamide)

Phenoxazinyl 6-hydroxynicotinamide was prepared according to the generalprocedure using 0.387 g (2.8 mmol) of 6-hydroxynicotinyl chloride and0.503 g (2.7 mmol) of phenoxazine to furnish 0.136 g (16%) of product.¹H NMR (DMSO-d₆, 400 MHz) d: 11.81 (bs, 1H), 7.62 (bs, 1H), 7.52 (bs,1H), 7.25 (m, 6H), 7.11 (m, 2H), 6.17 (d, J=10.4 Hz, 1H). IR (KBr) cm⁻¹:3445, 3070, 3050, 1645, 1615, 1480, 1345, 1265, 1115, 620. HREIMS m+/z(%): C₁₈H₁₂N₂O₃ (calc)=304.0849; C₁₈H₁₂N₂O₃ (obs)=304.0854 (100).

EXAMPLE 10 5-(N-(N-Methyl)piperazinyl)aminocarbonyl-2-pyridone (TautomerForm 1-Methylpiperazinyl 6-Hydroxynicotinamide)

1-Methylpiperazinyl 6-hydroxynicotinamide was prepared according to thegeneral procedure using 1.58 g (10.0 mmol) of 6-hydroxynicotinylchloride and 1.00 g (10.0 mmol) of 1-methylpiperazine to furnish 0.85 g(38%) of product. ¹H NMR (CDCl₃, 400 MHz) δ: 7.72 (d, J=3.1 Hz, 1H),7.60 (dd, J=3.1, 8.8 Hz, 1H), 6.59 (d, J=8.6 Hz, 1H), 3.67 (bt, J=4.5Hz, 4H), 2.47 (t, J=4.6 Hz, 4H), 2.35 (s, 3H). IR (CHCl₃) cm⁻¹: 3007,2946, 2803, 1681, 1660, 1632, 1614, 1459, 1435, 1297, 1275, 1137, 1000,731, 664. HREIMS m+/z (%): C₁₁H₁₅N₃O₂ (calc)=221.1161; C₁₁H₁₅N₃O₂(obs)=221.1163 (100).

Esterase Activity Assay

The esterase activity of BuChE or AChE was determined by a modificationof the method described by Ellman et al. (32), using a buffered5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) solution. Stock DTNBsolution consisted of 10 mM DTNB with 18 mM sodium bicarbonate in 0.1 Mphosphate buffer, pH 7.0. Working DTNB solution was prepared by mixing3.6 mL of 10 mM stock DTNB solution with 96.4 mL of 0.1 M phosphatebuffer at pH 8.0. The assay was carried out by mixing 2.7.mL of bufferedDTNB working solution (pH 8.0), 0.1 mL of BuChE or AChE in 0.005%aqueous gelatin (1 U/mL), and 0.1 mL of 50% aqueous acetonitrile, or asolution of a 2-pyridone compound of the invention in the same solvent,in a quartz cuvette of 1 cm path-length. Absorbance of this solution wascalibrated to zero and the reaction was commenced by adding 0.1 mL ofaqueous acetylthiocholine (AcSCh) or butyrylthiocholine (BuSCh)solutions of varying concentration (between 1.9 mM and 15 mM). The finalvolume was always 3.0 mL. The reactions were carried out at roomtemperature. The rate of change of absorbance (ΔA/min), reflecting therate of hydrolysis of BuSCh or AcSCh, was recorded every 5 seconds for atotal of 1 minute using a Milton-Roy uv-visible spectrophotometer set atλ=412 nm.

The slopes of Lineweaver-Burk plots versus Log of inhibitorconcentration were used to determine the inhibitor constant K_(i).

Trypsin Activity Assay

The effect of 2-pyridone compounds on trypsin-like enzymatic activityassociated with BuChE and that of human trypsin was determined usingNα-benzoyl-DL-arginine-p-nitroanilide (BAPNA) as the substrate. The sameprocedure was used to study the effect of the compounds on BuChE-trypsinmixture. Reactions were performed in 0.06 M Tris buffer at pH 8.0.Phosphate buffer was not used because BAPNA was found to undergobuffer-catalyzed hydrolysis in this medium at pH 8.0 over prolongedperiods. The incubations were carried out in 1.5 mL Eppendorf tubes bymixing 0.85 mL of 0.06 M Tris buffer (pH 8.0), 0.07 mL of up to 10 mMBAPNA, 0.03 mL of 50% aqueous acetonitrile, or a solution of 2-pyridonecompound (typically, a 5 mM working solution) in the same solvent, and0.05 mL of the enzyme solution (0.5-1.5 U of trypsin in 1 mMhydrochloric acid, or up to 5.0 U of BuChE in 0.005% aqueous gelatin).The final volume of the assay mixture was always 1.0 mL. The enzymereaction mixture was incubated at 40° C. for 15-45 hours. The use of 1.5U of trypsin gave an amount of p-nitroaniline (PNA) produced uponcleavage of BAPNA by trypsin after 22 hours of incubation that wassimilar to the amount of product formed by trypsin-like activityassociated with 5 U of BuChE under the same conditions.

The concentration of the PNA was determined by means of high performanceliquid chromatography (HPLC). This was carried out by injecting 20 μLsamples of the reaction mixture into a Waters system consisting of a 501pump, a 484 tunable uv-visible detector set at λ=380 nm to detect PNA,and a 745 data module. The column was a Nova-Pak C-18, 4μ cartridge (5mm×10 cm) in a RCM 8×10 Radial Pak cartridge holder. The solvent was 50%aqueous methanol at a flow rate of 1.5 mL/min. PNA is detected at 380 nmwhere BAPNA does not absorb. A standard curve was generated by injectingknown amounts of PNA into the HPLC system. At concentrations between0.03-1.0 nmol of PNA a linear relationship between the concentration ofthe product and the integrated area under the curve was observed. Therate of formation of PNA was calculated by using the following formula:

nmol of PNA /L/h=[integrated area under the curve×10⁵]÷[integrated areafor 1 nmol of PNA×time of incubation (h)].

TABLE 1 Pyridone compounds

Example R⁷R⁸N = X = 1

C═O 2

C═O 3

C═O 4

CH₂ 5

C═O 6

C═O 7

C═O 8

C═O 9

C═O 10

C═O

Esterase in Vitro Assay

The effect of increasing amount of exemplified 2-pyridone compounds(Examples 1 to 10) on the activity of human BuChE and AChE is shown inFIGS. 1 to 10.

The compounds of Examples 1 to 9 inhibited BuChE and AChE to varyingdegrees. Graphs of the slopes obtained from Lineweaver-Burk doublereciprocal graphs versus log of the concentration of each compound gavethe inhibition constant K_(i) for each compound. These values are shownin Table 2.

TABLE 2 Inhibition constants for 2-pyridone compounds of the invention.Esterase K_(i) (M) Pyridone compound BuChE(h) AChE(h) Example 1 3.75 ×10⁻⁵  2.6 × 10⁻⁴ Example 2 1.43 × 10⁻³ 1.31 × 10⁻⁴ Example 3 1.3 × 10⁻³ 7.6 × 10⁻³ Example 4 3.89 × 10⁻³ 4.77 × 10⁻³ Example 5 5.18 × 10⁻⁴Insignificant inhibition Example 6 4.51 × 10⁻³  1.3 × 10⁻² Example 71.37 × 10⁻⁴ Insignificant inhibition Example 8 4.29 × 10⁻⁵ Insignificantinhibition Example 9 4.29 × 10⁻⁵ Insignificant inhibition

Trypsin in Vitro Assay

The effect of two different exemplified compounds on the enzymaticactivity of the enzyme with trypsin-like activity associated with BuChEis shown in FIG. 11. The compounds of Examples 1 and 8 increased therate of hydrolysis of trypsin substrate compound BAPNA. The effect ofpyridone compounds on the enzymatic activity of trypsin itself is shownin FIG. 12. Example 8, used here as an example, substantially increasedthe hydrolytic activity of trypsin.

DISCUSSION Inhibition of Cholinesterases (Structure-activityRelationship)

The amide derivatives of 2-pyridone showed inhibitory activity towardscholinesterases. Some of the amides inhibited both AChE and BuChE to asimilar extent, while others inhibited one enzyme, primarily BuChE(Table 2), more than the other. One amine derivative5-(N,N-diethyl)aminomethyl-2-pyridone showed equal inhibitory activitytowards each cholinesterase studied.

It has been shown that the active site in cholinesterases is at thebottom of a “gorge” which is lined by aromatic amino acid residues, 12in AChE and 6 in BuChE. Some inhibitors bind to a peripheral site closeto the gorge to exert their action. In the case of the 2-pyridonederivatives of the present invention, the nature of inhibition is mixednon-competitive suggesting that these compounds most likely bind to theperipheral site near the active-site gorge. It is possible that thepyridone moiety binds at this site and the nitrogen containing sidechain binds to the amino acid residues in the gorge in a reversiblemanner. The difference in K_(i) values (Table 2) for the differentcompounds may be due to binding properties of the side chains.

Enhancement of the Activity of Serine Proteases

BuChE modifies the activity of trypsin by enhancing its activity undernormal conditions (30). This suggests that alteration of a synergisticeffect between BuChE and serine peptidases such as trypsin may play asignificant role in maturation of plaques because it has been shown thatcertain biochemical properties of BuChE are altered in AD. Certaincompounds of the present invention such as the phenothiazine-containingpyridone compound (Example 8) have also been found to enhance theactivity of trypsin. This enhancement is most likely through interactionof this molecule with trypsin at a peripheral site, which would changethe conformation of trypsin to facilitate hydrolysis of the substrate.

Other compounds of the present invention such as the dibenzyl compound(Example 1) do not have direct effect on trypsin alone. However, theactivity of the above-mentioned enzyme that has trypsin-like activityand which consistently co-purifies with BuChE, is considerably enhancedby the (dibenzyl compound (Example 1). This suggests that some compoundscan increase the activity of the trypsin-like protein by binding withBuChE such that the compound-BuChE complex, upon binding with thetrypsin-like protein, further facilitates the hydrolysis of thesubstrate.

Certain 2-pyridone compounds of the invention can inhibitcholinesterases. Some 2-pyridone compounds of the invention can modifythe activity of other serine hydrolases such as trypsin. These serinehydrolases are thought to be involved in APP processing. Because of theenhancement of the enzymatic activity of trypsin, the 2-pyridonescompounds of the present invention can be used to modify the progressionof AD by modifying APP processing, a step that is thought to be thecentral mechanism in the pathogenesis of AD.

Cholinesterases are not only involved in cholinergic neurotransmissionbut also in other biological processes such as development of thenervous system (33, 34). BuChE is found in high levels during neuroblastproliferation while AChE is found in high levels during neuronalmaturation (34). BuChE is found in high levels in certain tumors,particularly primary brain tumor such as gliomas. Because BuChE isinvolved in the process of cellular proliferation, the 2-pyridonecompounds of the present invention that are specific BuChE inhibitorscan be used to slow or stop growth of such brain tumors.

Glaucoma is one of the common eye diseases leading to blindness. Inglaucoma, there is increased intraocular pressure. Intraocular pressurecan be decreased by pupillary constriction. The pupil is innervated byboth sympathetic (adrenergic)and parasympathetic (cholinergic) nervoussystems. The parasympathetic nervous system, and cholinergic enhancingdrugs, cause pupillary constriction which can reduce intraocularpressure. The 2-pyridone compounds of the present is invention thatinhibit cholinesterases and raise acetylcholine am levels can be usedfor the treatment of ophthalmic diseases such as glaucoma.

The present invention extends to a pharmaceutical composition thatcomprises an active compound disclosed herein, or a pharmaceuticallyacceptable salt thereof, together with one or more pharmaceuticallyacceptable diluent or carriers, for modulating serine hydrolase activityin a mammal, preferably a human. The pharmaceutical composition can beused to treat, inhibit or prevent a pathological condition that ismanifested in an abnormal concentration of, and/or activity of, a serinehydrolase enzyme. Among those pathological conditions are Alzheimer'sdisease, tumours such as brain tumours, for example gliomas, andglaucoma.

Thus, the active compounds of the invention may be formulated for oral,buccal, transdermal (e.g., patch), intranasal, parenteral (e.g.,intravenous, intramuscular or subcutaneous), ophthalmic or rectaladministration or in a form suitable for administration by inhalation orinsufflation.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); filters (e.g., lactose, microcrystallinecellulose or calcium phosphate); lubricants (e.g., magnesium stearate,talc or silica); disintegrants (e.g., potato starch or sodium starchglycollate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, methyl cellulose or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters or ethyl alcohol); and preservatives(e.g., methyl or propyl p-hydroxybenzoates or sorbic acid).

For buccal administration the composition may take the form of tabletsof lozenges formulated in conventional manner.

The active compounds of the invention may be formulated for parenteraladministration by injection, including using conventionalcatheterization techniques or infusion. The active compounds of theinvention may also be formulated for topical ophthalmic administration.

Formulations for injection or topical ophthalmic administration may bepresented in unit dosage form, for example in ampules, or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulating agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for reconstitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The active compounds of the invention may also be formulated in rectalcompositions such as suppositories or retention enemas, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

For intranasal administration or administration by inhalation, theactive compounds of the invention are conveniently delivered in the formof a solution or suspension from a pump spray container that is squeezedor pumped by the patient. The compounds of the invention can also bedelivered in the form of an aerosol spray presentation from apressurized container or a nebulizer, with the use of a suitablepropellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. The pressurized containeror nebulizer may contain a solution or suspension of the activecompound. Capsules and cartridges (made, for example, from gelatin) foruse in an inhaler or insufflator may be formulated containing a powdermix of a compound of the invention and a suitable powder base such aslactose or starch.

As used herein, the term “effective amount” means an amount of acompound of the invention that is capable of inhibiting the symptoms ofa pathological condition described herein by modulation of serinehydrolase activity. The specific dose of a compound administeredaccording to this invention will be determined by the particularcircumstances surrounding the case including, for example, the compoundadministered, the route of administration, the state of being of thepatient, and the severity of the pathological condition. A proposed doseof an active compound of the invention for oral, parenteral, buccal ortopical ophthalmic administration to the average adult human for thetreatment of the conditions referred to above is 0.01 to 50 mg/kg of theactive ingredient per unit dose which could be administered, forexample, 1 to 4 times per day.

Aerosol formulations for treatment of the conditions referred to abovein the average adult human are preferably arranged so that each metereddose or “puff” of aerosol contains 20 μg to 1000 μg of the compound ofthe invention. The overall daily dose with an aerosol will be within therange a 100 μg to 10 mg. Administration may be several times daily, forexample 2, 3, 4 or 8 times, giving for example, 1, 2 or 3 doses eachtime.

All references cited herein are hereby incorporated by reference.

REFERENCES

1. Mayeux R., and Sano M. (1999) Treatment of Alzheimer's disease. NewEngland Journal of Medicine. 341: 1670-1679.

2. Patterson, C. J. S, Gauthier, S., Berman, H., Cohen, C. A., Feighter,J. W., Feldman, H. and Hogan, D. B. 1999. The recognition, assessmentand management of dementing disorders: conclusions from the CanadianConsensus Conference on Dementia. Canadian Medical Association Journal160: S1-S15.

3. Cummings, J. L., Vinters, H. V., Cole, G. M. and Khachaturian, Z. S.1998. Alzheimer's disease: Etiologies, pathophysiology, cognitivereserve, and treatment opportunities. Neurology 51: S2-S17.

4. Mirra, S. S., Heyman, A., McKeel, D., Sumi, S. M., Crain, B. J.,Brownlee, L. M., Vogel, F. S., Hughes, J. P., Van Belle, G., Berg, L.,and participating CERAD neuropathologists. 1991. The Consortium toEstablish a Registry for Alzheimer's Disease (CERAD). Part II.Standardization of the neuropathologic assessment of Alzheimer'sdisease. Neurology 41: 479-486.

5. Bartus, R. T., Dean, R. L., Beer, B., and Lippa, A. S. 1982. Thecholinergic hypothesis of geriatric memory dysfunction. Science 217:408-417.

6. Coyle, J. T., Price, D. L. and DeLong, M. R. 1983. Alzheimer'sdisease: A disorder of cortical cholinergic innervation. Science 219:1184-1190.

7. Geula, C., and Mesulam, M-M. (1995) Cholinesterases and the pathologyof Alzheimer disease. Alzheimer Disease and Associated Disorders 2:23-28.

8. Mesulam, M-M., and Geula, C. (1994) Butyrylcholinesterase reactivitydifferentiates the amyloid plaques of aging from those of dementia.Annals of Neurology. 36: 722-727.

9. Geula, C., and Mesulam, M-M. (1989) Special properties ofcholinesterases in the cerebral cortex of Alzheimer's disease. BrainResearch. 498: 185-189.

10. Greig, N. H., Lahiri, D. K., Soncrant, T. T., Utsuki, T., Yu, O. S.,Shaw, K. T. Y., Holloway, H. W., Myer, R. C., Wallace, W. C.,Haroutunian, V. and Ingram, D. K. 1998. Novel, selectivebutyrylcholinesterase (BChE) inhibitors for the treatment of Alzheimer'sdisease (AD). Society for Neuroscience Abstracts 24: 728.

11. Hardy, J. 1997 Amyloid, the presenilins and Alzheimer's disease.Trends in Neuroscience 20: 154-159.

12. Selkoe, D. J. 1991. The molecular pathology of Alzheimer's disease.Neuron 6: 487-498.

13. Guillozet, A. L., Smiley, J. F., Mash, D. C. and Mesulam, M-M. 1997.Butyrylcholinesterase in the life cycle of amyloid plaques. Annals ofNeurology 42: 909-918.

14. Kitaguchi, N., Takahashi, Y., Tokushima, Y., Shiojiri, S. and Ito,H. 1988. Novel precursor of Alzheimer's disease amyloid protein showsprotease inhibitory activity. Nature 331: 530-532.

15. Tanzi, R. E., McClatchey, A. I., Lamperti, E. D., Villa-Komaroff,L., Gusella, J. F. and Neve, R. L. 1988. Protease inhibitor domainencoded by an amyloid protein precursor mRNA associated with Alzheimer'sdisease. Nature 331:528-530.

16. Haas, C. and Selkoe, D. J. 1993. Cellular processing of β-amyloidprecursor protein and the genesis of amyloid β-peptide. Cell 75:1039-1042.

17. Hardy, J. 1997 Amyloid, the presenilins and Alzheimer's disease.Trends in Neuroscience 20: 154-159.

18. Esch, F. S., Keim, P. S., Beattie, E. C., Blacher, R. W., Culwell,A. R., Oltersdorf, T., McClure, D. and Ward, P. J. 1990. Cleavage ofamyloid peptide during constitutive processing of its precursor. Science248: 1122-1124.

19. Tomita, S., Kirino, Y. and Suzuki, T. 1998. A basic amino acid inthe cytoplasmic domain of Alzheimer's beta-amyloid precursor protein(APP) is essential for cleavage of APP at the alpha-site. Journal ofBiological Chemistry 273: 19304-19310.

20. Anderson, J. P., Esch, F. S., Keim, P. S., Sambamurti, K.,Lieberburg, I. and Robakis, N. K. 1991. Exact cleavage site of Alzheimeramyloid precursor in neuronal PC-12 cells. Neuroscience Letters 128:126-128.

21. Seubert, P., Vigo-Pelfrey, C., Esch, F., Lee, M., Dovey, H., Davis,D., Sinha, S., Schlossmacher, M., Whaley, J., Swindlhurst, C., McCormak,R., Wolfert, R., Selkoe, D. J., Lieberburg, I. and Schenk, D. 1992.Isolation and quantitation of soluble Alzheimer's β-peptide frombiological fluids. Nature 359: 325-327.

22. Hooper, N. M., Karran, E. H. and Turner, A. J. 1997. Membraneprotein secretases. Biochemical Journal 321: 265-279.

23. Worthington, V. 1993. Enzymes and related biochemicals. WorthingtonEnzyme Manual. Worthington Biochemical Corporation. Freehold, N.J.

24. Minn, A., Schubert, M., Neiss, W. F. and Muller-Hill, B. 1998.Enhanced GFAP expression in astrocytes of transgenic mice expressing thehuman brain-specific trypsinogen IV. Glia. 22: 338-347.

25. De Serres, M., Sherman, D., Chestnut, W., Merrill, B. M., Viveros,O. H. and Diliberto, E. J. Jr. (1993) Proteolysis at the secretase andamyloidogenic cleavage sites of the beta-amyloid precursor protein byacetyl cholinesterase and butyrylcholinesterase using model peptidesubstrates. Cell Molecular and Neurobiology 13: 279-287.

26. Meckelein, B., Marshall, D. C. L., Conn, K-J., Pietopaolo, M., VanNostrand, W. and Abraham, C. (1998) Identification of a novel serineprotease-like molecule in human brain. Brain Research Molecular BrainResearch 55: 181-197.

27. Wiegand, U., Corbach, S., Minn, A., Kang, J. and Muller-Hill, B.1993. Cloning of the cDNA encoding human brain trypsinogen andcharacterization of its product. Gene 136: 167-175.

28. Boopathy, R. and Balasubramanian, A. S. 1987. A peptidase activityexhibited by human serum pseudocholinesterase. European. Journal ofBiochemistry 162: 191-197.

29. Lockridge, O. 1982. Substance P hydrolysis by human serumcholinesterase. Journal of Neurochemistry 39: 106-110.

30. Darvesh, S., Kumar, R., Robert, S., Walsh, R. and Martin, E. (2000)Butyrylcholinesterase-Mediated Enhancement of the Enzymatic Activity ofTrypsin (In preparation).

31. Darvesh, S., Kumar, R. and Martin, E. (1999) Enzyme kinetics ofbutyrylcholinesterase and trypsin: Implications in Alzheimer's disease.Canadian Journal Neurological Sciences, 26, 546-547.

32. Ellman, G. L., Courtney, K. D., Andres, V. Jr. and Featherstone, R.M. (1961) A new and rapid calorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology, 7, 88-95.

33. Small, D. H., Michaelson, S. and Sberna, G. (1996) Non-classicalactions of cholinesterases: Role in cellular differentiation,tumorigenesis and Alzheimer's disease. Neurochemistry International, 28,453-483.

34. Layer, P. G. (1995) Non-classical roles of cholinesterases in theembryonic brain and possible links to Alzheimer disease AlzheimerDisease and Associated Disorders, 9, 29-36.

We claim:
 1. A compound of formula I:

or a pharmaceutically acceptable salt thereof; wherein X is C═O; R³, R⁴and R⁶ are each independently selected form the group consisting ofhydrogen, (C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl, (C₃-C₈)cycloalkyl,substituted (C₃-C₈)cycloalkyl, (C₂-C₁₂)alkenyl, substituted(C₂-C₁₂)alkenyl, (C₂-C₂)alkynyl, substituted (C₂-C₁₂)alkynyl,(C₆-C₁₄)aryl, substituted (C₆-C₁₄)aryl, (C₁-C₁₂)alkyl (C₆-C₁₄)aryl,substituted (C₁-C₁₂)alkyl (C₆-C₁₄)aryl, (C₆-C₁₄)aryl (C₁-C₁₂)alkyl,substituted (C₆-C₁₄)aryl (C₁-C₁₂)alkyl, (C₆-C₁₄)aryl (C₂-C₁₂)alkenyl,substituted (C₆-C₁₄)aryl (C₂-C₁₂)alkenyl, (C₆-C₁₄)aryl (C₂-C₁₂)alkynyl,substituted (C₆-C₁₄)aryl (C₂-C₁₂)alkynyl, trifluoromethyl, halogen,cyano and nitro; —S(O)R′, —S(O)₂R′, —S(O)₂OR′ and —S(O)₂NHR′, whereineach R′ is independently (C₁-C₁₂)alkyl, (C₂-C₁₂)alkenyl, (C₂-C₁₂)alkylor (C₆-C₁₄)aryl; —C(O)R″, wherein R″ is selected from the groupconsisting of hydrogen, (C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl,(C₃-C₈)cycloalkyl, substituted (C₃-C₈)cycloalkyl, (C₁-C₁₂)alkoxy,(C₁-C₁₂)alkylamino, (C₂-C₁₂)alkenyl, substituted (C₂-C₁₂)alkenyl,(C₂-C₁₂)alkynyl, substituted (C₂-C₁₂)alkynyl, (C₆-C₁₄)aryl, substituted(C₆-C₁₄)aryl, (C₆-C₁₄)aryloxy, (C₆-C₁₄)arylamino, (C₁-C₁₂)alkyl(C₆-C₁₄)aryl, substituted (C₁-C₁₂)alkyl (C₆-C₁₄)aryl, (C₆-C₁₄)aryl(C₁-C₁₂)alkyl, substituted (C₆-C₁₄)aryl (C₁-C₁₂)alkyl, (C₆-C₁₄)aryl(C₂-C₁₂)alkenyl, substituted (C₆-C₁₄)aryl (C₂-C₁₂)alkenyl, (C₆-C₁₄)aryl(C₂-C₁₂)alkynyl, substituted (C₆-C₁₄)aryl (C₂-C₁₂)alkynyl, andtrifluoromethyl; —OR′″ and —NR′″₂, wherein each R′″ is independentlyselected from hydrogen, (C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl,(C₃-C₈)cycloalkyl, substituted (C₃-C₈)cycloalkyl, (C₂-C₁₂)alkenyl,substituted (C₂-C₁₂)alkenyl, (C₂-C₁₂)alkynyl, substituted(C₂-C₁₂)alkynyl, (C₆-C₁₄)aryl, substituted (C₆-C₁₄)aryl, (C₁-C₁₂)alkyl(C₆-C₁₄)aryl, substituted (C₁-C₁₂)alkyl (C₆-C₁₄)aryl, (C₆-C₁₄)aryl(C₁-C₁₂)alkyl, substituted (C₆-C₁₄)aryl (C₁-C₁₂)alkyl, (C₆-C₁₄)aryl(C₂-C₁₂)alkenyl, substituted (C₆-C₁₄)aryl (C₂-C₁₂)alkenyl, (C₆-C₁₄)aryl(C₂-C₁₂)alkynyl, substituted (C₆-C₁₄)aryl (C₂-C₁₂)alkynyl,(C₆-C₁₄)aroyl, substituted (C₆-C₁₄)aroyl, (C₁-C₁₂)acyl andtrifluoromethyl; and —SR″″, wherein R″″ is selected from the groupconsisting of hydrogen, (C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl,(C₂-C₁₂)alkenyl, substituted (C₂-C₁₂)alkenyl, (C₂-C₁₂)alkynyl,substituted (C₂-C₁₂)alkynyl, (C₆-C₁₄)aryl, substituted (C₆-C₁₄)aryl,(C₁-C₁₂)alkyl (C₆-C₁₄)aryl, substituted (C₁-C₁₂)alkyl (C₆-C₁₄)aryl,(C₆-C₁₄)aryl (C₁-C₁₂)alkyl, substituted (C₆-C₁₄)aryl (C₁ -C₁₂)alkyl,(C₆-C₁₄)aryl (C₆-C₁₂)alkenyl, substituted (C₆-C₁₄)aryl (C₂-C₁₂)alkenyl,(C₆-C₁₄)aryl (C₂-C₁₂)alkynyl, substituted (C₆-C₁₄)aryl (C₂-C₁₂)alkynyl,and trifluoromethyl; and —NR⁷R⁸ together forms a (C₂-C₁₄)heterocyclic orsubstituted (C₂-C₁₄)heterocyclic; wherein the substituted groups listedabove are substituted with one or more substituents selected from thegroup consisting of hydroxy, methyl, (C₁-C₄)alkoxy, (C₆-C₁₄)aryl,(C₂-C₁₄)heterocyclic, halogen, trifluoromethyl, cyano, nitro, amino,carboxyl, carbamate, sulfonyl and sulfonamide; and the heterocyclicgroup contains at least one atom selected from oxygen, nitrogen andsulfur; with the proviso that —NR⁷R⁸ is other than imidazolyl when: (i)R³ is CN and R⁴ is H and R⁶ is methyl; (ii) R³, R⁴ and R⁶ are all H; or(iii) R³ and R⁴ are both H and R⁶ is methyl.
 2. The compound accordingto claim 1, wherein R³, R⁴ and R⁶ are each hydrogen.
 3. The compoundaccording to claim 1, wherein in the group —NR⁷R⁸, R⁷ and R⁸ togetherform a (C₂-C₇)alkylene group.
 4. The compound according to claim 2,wherein in the group —NR⁷R⁸, R⁷ and R⁸ together form a (C₂-C₇)alkylenegroup.
 5. The compound according to claim 1, wherein the compound is5-(1-pyrrolidinyl)carbonyl-2-pyridone.
 6. The compound according toclaim 1, wherein the compound is 5-(1-piperidinyl)carbonyl-2-pyridone.7. The compound according to claim 1, wherein the compound is5-(N-phenothiazinyl)carbonyl-2-pyridone.
 8. The compound according toclaim 1, wherein the compound is 5-(N-phenoxazinyl)carbonyl-2-pyridone.9. The compound according to claim 1, wherein the compound is5-(N-(N-methyl)piperazinyl)carbonyl-2-pyridone.
 10. A pharmaceuticalcomposition for inhibiting cholinesterase activity comprising a compoundof formula I:

or a pharmaceutically acceptable salt thereof, wherein X is C═O; R³, R⁴and R⁶ are each independently selected from the group consisting ofhydrogen, (C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl, (C₃-C₈)cycloalkyl,substituted (C₃-C₈)cycloalkyl, (C₂-C₁₂)alkenyl, substituted(C₂-C₁₂)alkenyl, (C₂-C₁₂)alkynyl, substituted (C₂-C₁₂)alkynyl,(C₆-C₁₄)aryl, substituted (C₆-C₁₄)aryl, (C₁-C₁₂)alkyl (C₆-C₁₄)aryl,substituted (C₁-C₁₂)alkyl (C₆-C₁₄)aryl, (C₆-C₁₄)aryl (C₁-C₁₂)alkyl,substituted (C₆-C₁₄)aryl (C₁-C₁₂)alkyl, (C₆-C₁₄)aryl (C₂-C₁₂)alkenyl,substituted (C₆-C₁₄)aryl (C₂-C₁₂)alkenyl, (C₆-C₁₄)aryl (C₂-C₁₂)alkynyl,substituted (C₆-C₁₄)aryl (C₂-C₁₂)alkynyl, trifluoromethyl, halogen,cyano and nitro; —S(O)R′, —S(O)₂R′, —S(O)₂OR′ and —S(O)₂NHR′, whereineach R′ is independently (C₁-C₁₂)alkyl, (C₂-C₁₂)alkenyl, (C₂-C₁₂)alkynylor (C₆-C₁₄)aryl; —C(O)R″, wherein R″ is selected from the groupconsisting of hydrogen, (C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl,(C₃-C₈)cycloalkyl, substituted (C₃-C₈)cycloalkyl, (C₁-C₁₂)alkoxy,(C₁-C₁₂)alkylamino, (C₂-C₁₂)alkenyl, substituted (C₂-C₁₂)alkenyl,(C₂-C₁₂)alkynyl, substituted (C₂-C₁₂)alkynyl, (C₆-C₁₄)aryl, substituted(C₆-C₁₄)aryl, (C₆-C₁₄)aryloxy, (C₆-C₁₄)arylamino, (C₁-C₁₂)alkyl(C₆-C₁₄)aryl, substituted (C₁-C₁₂)alkyl (C₆-C₁₄)aryl, (C₆-C₁₄)aryl(C₁-C₁₂)alkyl, substituted (C₆-C₁₄)aryl (C₁-C₁₂)alkyl, (C₆-C₁₄)aryl(C₂-C₁₂)alkenyl, substituted (C₆-C₁₄)aryl (C₂-C₁₂)alkenyl, (C₆-C₁₄)aryl(C₂-C₁₂)alkynyl, substituted (C₆-C₁₄)aryl (C₂-C₁₂)alkynyl, andtrifluoromethyl; and —OR′″ and —NR′″₂, wherein each R′″ is independentlyselected from hydrogen, (C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl,(C₃-C₈)cycloalkyl, substituted (C₃-C₈)cycloalkyl, (C₁-C₁₂)alkenyl,substituted (C₂-C₁₂)alkenyl, (C₂-C₁₂)alkynyl, substituted(C₂-C₁₂)alkynyl, (C₆-C₁₄)aryl, substituted (C₆-C₁₄)aryl, (C₁-C₁₂)alkyl(C₆-C₁₄)aryl, substituted (C₁-C₁₂)alkyl (C₆-C₁₄)aryl, (C₆-C₁₄)aryl(C₁-C₁₂)alkyl, substituted (C₆-C₁₄)aryl (C₁-C₁₂)alkyl, (C₆-C₁₄)aryl(C₂-C₁₂)alkenyl, substituted (C₆-C₁₄)aryl (C₂-C₁₂)alkenyl, (C₆-C₁₄)aryl(C₂-C₁₂)alkynyl, substituted (C₆-C₁₄)aryl (C₂-C₁₂)alkynyl,(C₆-C₁₄)aroyl, substituted (C₆-C₁₄)aroyl, (C₁-C₁₂)acyl andtrifluoromethyl; and —SR″″, wherein R″″ is selected from the groupconsisting of hydrogen, (C₁-C₁₂)alkyl, substituted (C₁-C₁₂)alkyl,(C₂-C₁₂)alkenyl, substituted (C₂-C₁₂)alkenyl, (C₂-C₁₂)alkynyl,substituted (C₂-C₁₂)alkynyl, (C₆-C₁₄)aryl, substituted (C₆-C₁₄)aryl,(C₁-C₁₂)alkyl (C₆-C₁₄)aryl, substituted (C₁-C₁₂)alkyl (C₆-C₁₄)aryl,(C₆-C₁₄)aryl (C₁-C₁₂)alkyl, substituted (C₆-C₁₄)aryl (C₁-C₁₂)alkyl,(C₆-C₁₄)aryl (C₂-C₁₂)alkenyl, substituted (C₆-C₄)aryl (C₂-C₁₂)alkenyl,(C₆-C₁₄)aryl (C₂-C₁₂)alkynyl, substituted (C₆-C₁₄)aryl (C₂-C₁₂)alkynyl,and trifluoromethyl; and —NR⁷R⁸ together forms a (C₂-C₁₄)heterocyclic orsubstituted (C₂-C₁₄)heterocyclic; wherein the substituted groups listedabove are substituted with one or more substituents selected from thegroup consisting of hydroxy, methyl, (C₁-C₄)alkoxy, (C₆-C₁₄)aryl,(C₂-C₁₄)heterocyclic, halogen, trifluoromethyl, cyano, nitro, amino,carboxyl, carbamate, sulfonyl and sulfonamide; and the heterocyclicgroup contains at least one atom selected from oxygen, nitrogen andsulfur; with the proviso that —NR⁷R⁸ is other than imidazolyl when: (i)R³ is CN and R⁴ is H and R⁶ is methyl; (ii) R³, R⁴ and R⁶ are all H; or(iii) R³ and R⁴ are both H and R⁶ is methyl; together with apharmaceutically acceptable diluent or carrier.
 11. The pharmaceuticalcomposition according to claim 10, wherein the cholinesterase isbutyrylcholinesterase (BuChE).
 12. The pharmaceutical compositionaccording to claim 10, wherein the cholinesterase isacetylcholinesterase (AChE).
 13. The pharmaceutical compositionaccording to claim 11, for the treatment of Alzheimer's disease.
 14. Thepharmaceutical composition according to claim 11, for the treatment of aprimary brain tumour.
 15. The pharmaceutical composition according toclaim 14, wherein the primary brain tumour is a glioma.
 16. Thepharmaceutical composition according to claim 11, for the treatment ofglaucoma.
 17. The compound according to claim 2, wherein —NR⁷R⁸ togetherforms a (C₂-C₁₄)heterocyclic or substituted (C₂-C₁₄)heterocyclic whichcomprises one or more fused benzo groups.